Although several reports have been published on recombinant protein expression using Drosophila cells, information on their metabolism and growth in vitro is relatively scarce. In the present study, we have analyzed the growth and metabolism of transfected S2 cells (S2AcRVGP) in bioreactor cultures with serum-free medium Sf900 II, to evaluate its potential for mass production of a rabies virus glycoprotein (RVGP). Cells were cultured in a 3 l-stirred-tank bioreactor at 28 degrees C with pH controlled at 6.2 and dissolved oxygen at 50% air saturation. The cells attained a specific growth rate and maximum cell density as high as 0.084 h(-1) and 2.3 x 10(7 )cell ml(-1), respectively. The main substrates consumed during this rapid growth phase were glucose, glutamine and proline. An atypical accumulation of ammonia and alanine was observed in the culture medium, up to 62 mM and 47 mM, respectively, but lactate was produced in low levels. After exhaustion of glutamine and proline as energy sources, alanine was consumed and production of ammonia increased. The production of recombinant RVGP reached concentrations as high as 178 mug l(-1). Premature exhaustion of glutamine, serine and cysteine could be related to degradation of the recombinant glycoprotein. In general, the results demonstrated that S2AcRVGP can be considered an effective vehicle for large-scale recombinant glycoprotein expression and that several critical factors of the bioprocess could be optimized to increase the quality and productivity of the RVGP.